It is often desirable to detect specific DNA sequences in a mixture of sequences or in a sample that contains DNA and other ingredients. For instance, detection of specific DNA sequences is often used to determine whether a particular bacterium is present in a biological sample. Many techniques can be used including cycling assays such as polymerase chain reaction (PCR) and exonuclease cycling assays, in which exonucleases are used to digest probes. Lambda exonuclease has been used in an exonuclease cycling assay to digest double stranded DNA (dsDNA) from its 5′ end (Copley and Boot, Exonuclease Cycling Assay: An Amplified Assay for the Detection of Specific DNA Sequences, Biotechniques 1992 December; 13(6):888-92). Lambda exonuclease has very little activity on a single stranded DNA (ssDNA). Digestion by lambda exonuclease requires a phosphate at the 5′ end of the DNA strand to be digested. The 5′ exonuclease activity of a DNA polymerase (Taq polymerase) is used for real time PCR analysis. This assay requires both DNA synthesis and probe digestion by the exonuclease activity of the polymerase (U.S. Pat. No. 5,716,784). It also requires a probe with a blocked 3′ end. The 3′ exonuclease activity of exonuclease III has been used for a cycling reaction (Okano and Kambara, DNA Probe Assay Based on Exonuclease III Digestion of Probes Hybridized on Target DNA, Analytical Biochem 1995; 288, 101-8), but the background is extremely high.
The exonuclease cycling assay has a number of drawbacks. First, the assay tends to have high background activity. Second, both probe and target DNA can be degraded preventing a cycling assay where a probe binds and is degraded in a cycling reaction. Third, the enzyme requires a 5′ phosphate. This limits the position and type of modifications that can be made to the probe. Thus, there remains a need for a reliable and sensitive exonuclease cycling assay to detect specific nucleotide sequences.